RESUMEN
Glioblastoma multiforme (GBM) is the most aggressive type of glioma, displaying atypical glycosylation pattern that may modulate signaling pathways involved in tumorigenesis. Lectins are glycan binding proteins with antitumor properties. The present study was designed to evaluate the antitumor capacity of the Dioclea reflexa lectin (DrfL) on glioma cell cultures. Our results demonstrated that DrfL induced morphological changes and cytotoxic effects in glioma cell cultures of C6, U-87MG and GBM1 cell lines. The action of DrfL was dependent upon interaction with glycans, and required a carbohydrate recognition domain (CRD), and the cytotoxic effect was apparently selective for tumor cells, not altering viability and morphology of primary astrocytes. DrfL inhibited tumor cell migration, adhesion, proliferation and survival, and these effects were accompanied by activation of p38MAPK and JNK (p46/54), along with inhibition of Akt and ERK1/2. DrfL also upregulated pro-apoptotic (BNIP3 and PUMA) and autophagic proteins (Atg5 and LC3 cleavage) in GBM cells. Noteworthy, inhibition of autophagy and caspase-8 were both able to attenuate cell death in GBM cells treated with DrfL. Our results indicate that DrfL cytotoxicity against GBM involves modulation of cell pathways, including MAPKs and Akt, which are associated with autophagy and caspase-8 dependent cell death.
Asunto(s)
Antineoplásicos , Muerte Celular Autofágica , Dioclea , Glioma , Humanos , Dioclea/química , Caspasa 8/metabolismo , Caspasa 8/farmacología , Caspasa 8/uso terapéutico , Lectinas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/farmacología , Proteínas Proto-Oncogénicas c-akt/uso terapéutico , Línea Celular Tumoral , Glioma/tratamiento farmacológico , Glioma/metabolismo , Glioma/patología , Movimiento Celular , Autofagia , Antineoplásicos/farmacología , Proliferación Celular , ApoptosisRESUMEN
BRCA-1 is a nuclear protein involved in DNA repair, transcriptional regulation, and cell cycle control. Its involvement in other cellular processes has been described. Here, we aimed to investigate the role of BRCA-1 in macrophages M(LPS), M(IL-4), and tumor cell-induced differentiation. We used siRNAs to knockdown BRCA-1 in RAW 264.7 macrophages exposed to LPS, IL-4, and C6 glioma cells conditioned medium (CMC6), and evaluated macrophage differentiation markers and functional phagocytic activity as well as DNA damage and cell survival in the presence and absence of BRCA-1. LPS and CMC6, but not by IL-4, increased DNA damage in macrophages, and this effect was more pronounced in BRCA-1-depleted cells, including M(IL-4). BRCA-1 depletion impaired expression of pro-inflammatory cytokines, TNF-α and IL-6, and reduced the phagocytic activity of macrophages in response to LPS. In CMC6-induced differentiation, BRCA-1 knockdown inhibited TNF-α and IL-6 expression which was accompanied by upregulation of the anti-inflammatory markers IL-10 and TGF-ß and reduced phagocytosis. In contrast, M(IL-4) phenotype was not affected by BRCA-1 status. Molecular docking predicted that the conserved BRCA-1 domain BRCT can interact with the p65 subunit of NF-κB. Immunofluorescence assays showed that BRCA-1 and p65 co-localize in the nucleus of LPS-treated macrophages and reporter gene assay showed that depletion of BRCA-1 decreased LPS and CMC6-induced NF-κB transactivation. IL-4 had no effect upon NF-κB. Taken together, our findings suggest a role of BRCA-1 in macrophage differentiation and phagocytosis induced by LPS and tumor cells secretoma, but not IL-4, in a mechanism associated with inhibition of NF-κB.
Asunto(s)
Proteína BRCA1/metabolismo , Polaridad Celular , Inflamación/patología , Activación de Macrófagos , Macrófagos/metabolismo , Macrófagos/patología , FN-kappa B/metabolismo , Animales , Biomarcadores/metabolismo , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Polaridad Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Daño del ADN , Inflamación/metabolismo , Lipopolisacáridos , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Metaloproteinasas de la Matriz/metabolismo , Ratones , Fagocitosis/efectos de los fármacos , Células RAW 264.7 , ARN Interferente Pequeño/metabolismo , RatasRESUMEN
BACKGROUND/AIMS: Heat shock protein 70 (HSP70) has been recently described with extracellular actions, where it is actively released in inflammatory conditions. Acting as DAMPs (damage associated molecular pattern), extracellular HSP70 (eHSP70) interacts with membrane receptors and activates inflammatory pathways. At this context, the receptor for advanced glycation endproducts (RAGE) emerges as a possible candidate for interaction with eHSP70. RAGE is a pattern-recognition receptor and its expression is increased in several diseases related to a chronic pro-inflammatory state. One of the main consequences of RAGE ligand-binding is the ERK1/2 (extracellular signal-regulated kinases)-dependent activation of NF-kB (nuclear factor kappa B), which leads to expression of TNF-α (tumor necrosis factor alpha) and other cytokines. The purpose of this work is to elucidate if eHSP70 is able to evoke RAGE-dependent signaling using A549 human lung cancer cells, which constitutively express RAGE. METHODS: Immunoprecipitation and protein proximity assay were utilized to demonstrate the linkage between RAGE and eHSP70. To investigate RAGE relevance on cell response to eHSP70, siRNA was used to knockdown the receptor expression. Signaling pathways activation were evaluated by western blotting, gene reporter luciferase and real time quantitative PCR. RESULTS: Protein eHSP70 shown to be interacting physically with the receptor RAGE in our cell model. Treatment with eHSP70 caused ERK1/2 activation and NF-κB transactivation impaired by RAGE knockdown. Moreover, the stimulation of pro-inflammatory cytokines expression by eHSP70 was inhibited in RAGE-silenced cells. Finally, conditioned medium of eHSP70-treated A549 cells caused differential effects in monocytes cytokine expression when A549 RAGE expression is inhibited. CONCLUSIONS: Our results evidence eHSP70 as a novel RAGE agonist capable of influence the cross-talk between cancer and immune system cells.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Células A549 , Citocinas/genética , Citocinas/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/farmacología , Humanos , Immunoblotting , Inmunoprecipitación , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , FN-kappa B/genética , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor para Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Receptor para Productos Finales de Glicación Avanzada/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Activación Transcripcional , Células U937RESUMEN
Glioma cells release cytokines to stimulate inflammation that facilitates cell proliferation. Here, we show that Lipopolysaccharide (LPS) treatment could induce glioma cells to proliferate and this process was dependent on nucleotide receptor activation as well as interleukin-8 (IL-8/CXCL8) secretion. We observed that extracellular nucleotides controlled IL-8/CXCL8 and monocyte chemoattractant protein 1 (MCP-1/CCL2) release by U251MG and U87MG human glioma cell lines via P2X7 and P2Y6 receptor activation. The LPS-induced release of these cytokines was also modulated by purinergic receptor activation since IL-8 and MCP-1 release was decreased by the nucleotide scavenger apyrase as well as by the pharmacological P2Y6 receptor antagonists suramin and MRS2578. In agreement with these observations, the knockdown of P2Y6 expression decreased LPS-induced IL-8 release as well as the spontaneous release of IL-8 and MCP-1, suggesting an endogenous basal release of nucleotides. Moreover, high millimolar concentrations of ATP increased IL-8 and MCP-1 release by the glioma cells stimulated with suboptimal LPS concentration which were blocked by P2X7 and P2Y6 antagonists. Altogether, these data suggest that extracellular nucleotides control glioma growth via P2 receptor-dependent IL-8 and MCP-1 secretions.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Proliferación Celular , Quimiocina CCL2/metabolismo , Glioma/metabolismo , Interleucina-8/metabolismo , Receptores Purinérgicos/fisiología , Secuencia de Bases , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Cartilla de ADN , Glioma/patología , Humanos , Reacción en Cadena de la Polimerasa , Receptores Purinérgicos/genética , Receptores Purinérgicos/metabolismo , Transducción de SeñalRESUMEN
Tropospheric ozone (O3) background concentrations have increased since pre-industrial times, reaching phytotoxic concentrations in many regions globally. However, the effect of high O3 concentrations on quality of fruit and vegetables remains unknown. Here, we evaluated whether O3 pollution alters the quality of Capsicum baccatum peppers by changing the secondary compound profiles and biological activity of the fruit. C. baccatum pepper plants were exposed to ozone for 62 days in an open-top chamber at a mean O3 concentration of 171.6µg/m(3). Capsaicin levels decreased by 50% in the pericarp, but remained unchanged in the seeds. In contrast, the total carotenoid content increased by 52.8% in the pericarp. The content of total phenolic compounds increased by 17% in the pericarp. The total antioxidant potential decreased by 87% in seeds of O3-treated plants. The seeds contributed more than the pericarp to the total radical-trapping antioxidant potential and total antioxidant reactivity. O3 treatment impaired the ferric-reducing antioxidant power of the seeds and reduced NO(â¢)-scavenging activity in the pericarp. However, O3 treatment increased ferrous ion-chelating activity and hydroxyl radical-scavenging activity in the pericarp. Our results confirm that O3 alters the secondary metabolite profile of C. baccatum pepper fruits and, consequently, their biological activity profile.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Capsicum/efectos de los fármacos , Oxidantes/toxicidad , Ozono/toxicidad , Antiinflamatorios/metabolismo , Antioxidantes/metabolismo , Capsaicina/metabolismo , Capsicum/metabolismo , Carotenoides/metabolismo , Frutas/efectos de los fármacos , Frutas/metabolismo , Fenoles/metabolismo , Semillas/efectos de los fármacos , Semillas/metabolismoRESUMEN
Auranofin is a thioredoxin reductase-1 inhibitor originally approved for the treatment of rheumatoid arthritis. Recently, auranofin has been repurposed as an anticancer drug, with pharmacological activity reported in multiple cancer types. In this study, we characterized transcriptional and genetic alterations associated with auranofin response in cancer. By integrating data from an auranofin cytotoxicity screen with transcriptome profiling of lung cancer cell lines, we identified an auranofin resistance signature comprising 29 genes, most of which are classical targets of the transcription factor NRF2, such as genes involved in glutathione metabolism (GCLC, GSR, SLC7A11) and thioredoxin system (TXN, TXNRD1). Pan-cancer analysis revealed that mutations in NRF2 pathway genes, namely KEAP1 and NFE2L2, are strongly associated with overexpression of the auranofin resistance gene set. By clustering cancer types based on auranofin resistance signature expression, hepatocellular carcinoma, and a subset of non-small cell lung cancer, head-neck squamous cell carcinoma, and esophageal cancer carrying NFE2L2/KEAP1 mutations were predicted resistant, whereas leukemia, lymphoma, and multiple myeloma were predicted sensitive to auranofin. Cell viability assays in a panel of 20 cancer cell lines confirmed the augmented sensitivity of hematological cancers to auranofin; an effect associated with dependence upon glutathione and decreased expression of NRF2 target genes involved in GSH synthesis and recycling (GCLC, GCLM and GSR) in these cancer types. In summary, the omics-based identification of sensitive/resistant cancers and genetic alterations associated with these phenotypes may guide an appropriate repurposing of auranofin in cancer therapy.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Auranofina/farmacología , Auranofina/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Reposicionamiento de Medicamentos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/uso terapéutico , Glutatión/metabolismo , Glutatión/uso terapéuticoRESUMEN
Peripheral neuropathy is an important adverse effect caused by some chemotherapeutic agents, including oxaliplatin (OXA). OXA-induced peripheral neuropathy (OIPN) is a challenging condition due to diagnostic complexities and a lack of effective treatment. In this study, we investigated the antiallodynic effect of ß-caryophyllene (BCP), a cannabinoid type 2 (CB2) receptor agonist, in a mouse model of OIPN. BCP treatment inhibited OXA-induced mechanical and cold allodynia in both preventive and therapeutic drug treatment regimens. Experiments with the CB2 receptor agonist GW405833 confirmed the role of CB2 receptors in OIPN. The CB2 antagonist SR144528 abrogated the anti-nociceptive effect of BCP on mechanical allodynia, without impacting OXA-induced sensitivity to cold. BCP decreased neuroinflammation, as inferred from TNF, IL-1ß, IL-6, and IL-10 profiling, and also reduced ROS production, lipid peroxidation, and 4-hydroxynonenal protein adduct formation in the spinal cords of OXA-treated mice. BCP did not affect the antitumor response to OXA or its impact on blood cell counts, implying that the cytotoxicity of OXA was preserved. These results underscore BCP as a candidate drug for OIPN treatment via CB2 receptor-dependent mechanisms, and anti-inflammatory and antioxidant responses in the spinal cord.
RESUMEN
In sepsis, toll-like receptor (TLR)-4 modulates the migration of neutrophils to infectious foci, favoring bacteremia and mortality. In experimental sepsis, organ dysfunction and cytokines released by activated macrophages can be reduced by gastrin-releasing peptide (GRP) receptor (GRPR) antagonist RC-3095. Here we report a link between GRPR and TLR-4 in experimental models and in sepsis patients. RAW 264.7 culture cells were exposed to lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-α and RC-3095 (10 ng/mL). Male Wistar rats were subjected to cecal ligation and puncture (CLP), and RC-3095 was administered (3 mg/kg, subcutaneously); after 6 h, we removed the blood, bronchoalveolar lavage, peritoneal lavage and lung. Human patients with a clinical diagnosis of sepsis received a continuous infusion with RC-3095 (3 mg/kg, intravenous) over a period of 12 h, and plasma was collected before and after RC-3095 administration and, in a different set of patients with systemic inflammatory response syndrome (SIRS) or sepsis, GRP plasma levels were determined. RC-3095 inhibited TLR-4, extracellular-signal-related kinase (ERK)-1/2, Jun NH(2)-terminal kinase (JNK) and Akt and decreased activation of activator protein 1 (AP-1), nuclear factor (NF)-κB and interleukin (IL)-6 in macrophages stimulated by LPS. It also decreased IL-6 release from macrophages stimulated by TNF-α. RC-3095 treatment in CLP rats decreased lung TLR-4, reduced the migration of cells to the lung and reduced systemic cytokines and bacterial dissemination. Patients with sepsis and systemic inflammatory response syndrome have elevated plasma levels of GRP, which associates with clinical outcome in the sepsis patients. These findings highlight the role of GRPR signaling in sepsis outcome and the beneficial action of GRPR antagonists in controlling the inflammatory response in sepsis through a mechanism involving at least inhibition of TLR-4 signaling.
Asunto(s)
Receptores de Bombesina/antagonistas & inhibidores , Sepsis/prevención & control , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Adulto , Animales , Bombesina/administración & dosificación , Bombesina/análogos & derivados , Bombesina/farmacología , Movimiento Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Péptido Liberador de Gastrina/sangre , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-6/sangre , Lipopolisacáridos/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Modelos Biológicos , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores de Bombesina/metabolismo , Sepsis/sangre , Sepsis/metabolismo , Sepsis/microbiología , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
Proteasome inhibitors are emerging as a new class of anticancer agents. In this work, we examined the mechanisms underlying cytotoxicity, selectivity and adjuvant potential of the proteasome inhibitor MG132 in a panel of glioblastoma (GBM) cells (U138MG, C6, U87 and U373) and in normal astrocytes. MG132 markedly inhibited GBM cells growth irrespective of the p53 or PTEN mutational status of the cells whereas astrocytic viability was not affected, suggesting a selective toxicity of MG132 to cancerous glial cells. Mechanistically, MG132 arrested cells in G2/M phase of the cell cycle and increased p21(WAF1) protein immunocontent. Following cell arrest, cells become apoptotic as shown by annexin-V binding, caspase-3 activation, chromatin condensation and formation of sub-G1 apoptotic cells. MG132 promoted mitochondrial depolarization and decreased the mitochondrial antiapoptotic protein bcl-xL; it also induced activation of JNK and p38, and inhibition of NFkappaB and PI3K/Akt survival pathways. Pre-treatment of GBMs with the mitochondrial permeability transition pore inhibitor, bongkrekic acid, or pharmacological inhibitors of JNK1/2 and p38, SP600125 and SB203580, attenuated MG132-induced cell death. Besides its apoptotic effect alone, MG132 also enhanced the antiglioma effect of the chemotherapeutics cisplatin, taxol and doxorubicin in C6 and U138MG cells, indicating an adjuvant/chemosensitizer potential. In summary, MG132 exerted profound and selective toxicity in GBMs, being a potential agent for further testing in animal models of the disease.
Asunto(s)
Adyuvantes Farmacéuticos/administración & dosificación , Antineoplásicos/administración & dosificación , Leupeptinas/administración & dosificación , Inhibidores de Proteasoma/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/administración & dosificación , Doxorrubicina/administración & dosificación , Glioblastoma , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/antagonistas & inhibidores , Paclitaxel/administración & dosificación , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Zika virus (ZIKV) is a single-strand RNA mosquito-borne flavivirus with significant public health impact. ZIKV infection induces double-strand DNA breaks (DSBs) in human neural progenitor cells that may contribute to severe neuronal manifestations in newborns. The DNA-PK complex plays a critical role in repairing DSBs and in the innate immune response to infection. It is unknown, however, whether DNA-PK regulates ZIKV infection. Here we investigated the role of DNA-PKcs, the catalytic subunit of DNA-PK, during ZIKV infection. We demonstrate that DNA-PKcs restricts the spread of ZIKV infection in human epithelial cells. Increased ZIKV replication and spread in DNA-PKcs deficient cells is related to a notable decrease in transcription of type I and III interferons as well as IFIT1, IFIT2, and IL6. This was shown to be independent of IRF1, IRF3, or p65, canonical transcription factors necessary for activation of both type I and III interferon promoters. The mechanism of DNA-PKcs to restrict ZIKV infection is independent of DSB. Thus, these data suggest a non-canonical role for DNA-PK during Zika virus infection, acting downstream of IFNs transcription factors for an efficient antiviral immune response.
Asunto(s)
Infección por el Virus Zika , Virus Zika , Recién Nacido , Animales , Humanos , Virus Zika/fisiología , Replicación Viral , Interferones/farmacología , Antivirales/uso terapéutico , ADNRESUMEN
Though, it is quite well-known how retinoic acid (RA) is able to induce neuritogenesis in different in vitro models, the putative role exerted by reactive oxygen species (ROS) during this process still need to be further studied. For such purpose, we used a neuronal-like cell line (SH-SY5Y cells) in order to investigate whether the antioxidant Trolox (a hydrophilic analog of alpha-tocopherol) could have any effect on the number of RA-induced neurites, and how significant changes in cellular redox homeostasis may affect the cellular endogenous expression of tyrosine hydroxylase (TH). Our results show a significant enhancement of RA (10 µM)-induced neuritogenesis and TH endogenous expression, when cells were co-treated with Trolox (100 µM) for 7 days. Moreover, this effect was associated with an improvement in cellular viability. The mechanism seems to mainly involve PI3 K/Akt rather than MEK signaling pathway. Therefore, our data demonstrate that concomitant decreases in basal reactive oxygen species (ROS) production could exert a positive effect on the neuritogenic process of RA-treated SH-SY5Y cells.
Asunto(s)
Antioxidantes/farmacología , Cromanos/farmacología , Neuritas/metabolismo , Neuroblastoma/enzimología , Neurogénesis/efectos de los fármacos , Tretinoina/farmacología , Tirosina 3-Monooxigenasa/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Peroxidación de Lípido/efectos de los fármacos , Microscopía de Contraste de Fase , Neuritas/efectos de los fármacos , Neuroblastoma/patología , Estrés Oxidativo/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive loss of the nigrostriatal dopaminergic neurons that are associated with motor alterations and non-motor manifestations (such as depression). Neuroinflammation is a process with a critical role in the pathogenesis of PD. In this regard, toll-like receptor 4 (TLR4) is a central mediator of immune response in PD. Moreover, there are gender-related differences in the incidence, prevalence, and clinical features of PD. Therefore, we aimed to elucidate the role of TLR4 in the sex-dependent response to dopaminergic denervation induced by 6-hydroxydopamine (6-OHDA) in mice. Female and male adult wildtype (WT) and TLR4 knockout (TLR4-/-) mice were administered with unilateral injection of 6-OHDA in the dorsal striatum, and non-motor and motor impairments were evaluated for 30 days, followed by biochemistry analysis in the substantia nigra pars compacta (SNc), dorsal striatum, and dorsoventral cortex. Early non-motor impairments (i.e., depressive-like behavior and spatial learning deficits) induced by 6-OHDA were observed in the male WT mice but not in male TLR4-/- or female mice. Motor alterations were observed after administration of 6-OHDA in both strains, and the lack of TLR4 was also related to motor commitment. Moreover, ablation of TLR4 prevented 6-OHDA-induced dopaminergic denervation and microgliosis in the SNc, selectively in female mice. These results reinforced the existence of sex-biased alterations in PD and indicated TLR4 as a promising therapeutic target for the motor and non-motor symptoms of PD, which will help counteract the neuroinflammatory and neurodegenerative processes.
Asunto(s)
Encéfalo/efectos de los fármacos , Enfermedad de Parkinson/tratamiento farmacológico , Factores Sexuales , Receptor Toll-Like 4/metabolismo , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Femenino , Hidroxidopaminas/farmacología , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Degeneración Nerviosa/inducido químicamente , Enfermedades Neurodegenerativas/inducido químicamente , Enfermedades Neurodegenerativas/patología , Enfermedad de Parkinson/genética , Receptor Toll-Like 4/efectos de los fármacos , Receptor Toll-Like 4/genéticaRESUMEN
Activating mutations in the KEAP1/NRF2 pathway characterize a subset of non-small cell lung cancer (NSCLC) associated with chemoresistance and poor prognosis. We herein evaluated the relationship between 64 oxidative stress-related genes and overall survival data from 35 lung cancer datasets. Thioredoxin reductase-1 (TXNRD1) stood out as the most significant predictor of poor outcome. In a cohort of NSCLC patients, high TXNRD1 protein levels correlated with shorter disease-free survival and distal metastasis-free survival post-surgery, including a subset of individuals treated with platinum-based adjuvant chemotherapy. Bioinformatics analysis revealed that NSCLC tumors harboring genetic alterations in the NRF2 pathway (KEAP1, NFE2L2 and CUL3 mutations, and NFE2L2 amplification) overexpress TXNRD1, while no association with EGFR, KRAS, TP53 and PIK3CA mutations was found. In addition, nuclear accumulation of NRF2 overlapped with upregulated TXNRD1 protein in NSCLC tumors. Functional cell assays and gene dependency analysis revealed that NRF2, but not TXNRD1, has a pivotal role in KEAP1 mutant cells' survival. KEAP1 mutants overexpress TXNRD1 and are less susceptible to the cytotoxic effects of the TXNRD1 inhibitor auranofin when compared to wild-type cell lines. Inhibition of NRF2 with siRNA or ML-385, and glutathione depletion with buthionine-sulfoximine, sensitized KEAP1 mutant A549 cells to auranofin. NRF2 knockdown and GSH depletion also augmented cisplatin cytotoxicity in A549 cells, whereas auranofin had no effect. In summary, these findings suggest that TXNRD1 is not a key determinant of malignant phenotypes in KEAP1 mutant cells, although this protein can be a surrogate marker of NRF2 pathway activation, predicting tumor recurrence and possibly other aggressive phenotypes associated with NRF2 hyperactivation in NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Tiorredoxina Reductasa 1 , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Proteínas Cullin , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Recurrencia Local de Neoplasia/genética , Transducción de Señal , Tiorredoxina Reductasa 1/genética , Tiorredoxina Reductasa 1/metabolismoRESUMEN
Chemotherapy-Induced Peripheral Neuropathy (CIPN) is a common, difficult-to-treat, and dose-limiting side effect associated with Oxaliplatin (OXA) treatment. In this study, we evaluated the effect of three antioxidants - namely N-acetylcysteine, α-lipoic acid and vitamin E - upon nociceptive parameters and antitumor efficacy of OXA in a tumor-bearing Swiss mice model. Oral treatment with antioxidants inhibited both mechanical and cold allodynia when concomitantly administrated with OXA (preventive protocol), as well as in animals with previously established CIPN (therapeutic protocol). OXA increased Reactive Oxygen Species (ROS) production and lipoperoxidation, and augmented the content of pro-inflammatory cytokines (IL-1ß and TNF-α) and expression of the astrocytic marker Gfap mRNA in the spinal cord. Antioxidants decreased ROS production and lipoperoxidation, and abolished neuroinflammation in OXA-treated animals. Toll-like receptor 4 (Tlr4) and inflammasome enzyme caspase-1/11 knockout mice treated with OXA showed reduced levels of pro-inflammatory cytokines (but not oxidative stress) in the spinal cord, which were associated with resistance to OXA-induced mechanical allodynia. Lastly, antioxidants affected neither antitumor activity nor hematological toxicity of OXA in vivo. The herein presented results are provocative for further evaluation of antioxidants in clinical management of chemotherapy-induced peripheral neuropathy. PERSPECTIVE: This study reports preventive and therapeutic efficacy of orally administrated antioxidants (N-acetylcysteine, α-lipoic-acid and Vitamin-E) in alleviating oxaliplatin-induced peripheral neuropathy in tumor-bearing mice. Antioxidants' anti-nociceptive effects are associated with inhibition of ROS-dependent neuroinflammation, and occur at no detriment of OXA antitumor activity, therefore indicating a translational potential of these compounds.
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Antineoplásicos/efectos adversos , Antioxidantes/farmacología , Hiperalgesia , Neoplasias/tratamiento farmacológico , Enfermedades Neuroinflamatorias , Oxaliplatino/efectos adversos , Estrés Oxidativo/efectos de los fármacos , Enfermedades del Sistema Nervioso Periférico , Médula Espinal , Animales , Modelos Animales de Enfermedad , Hiperalgesia/inducido químicamente , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedades Neuroinflamatorias/inducido químicamente , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/inmunología , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/prevención & control , Médula Espinal/efectos de los fármacos , Médula Espinal/inmunología , Médula Espinal/metabolismo , Receptor Toll-Like 4RESUMEN
Obesity is a major risk factor for breast cancer, especially in post-menopausal women. In the breast tissue of obese women, cyclooxygenase-2 (COX-2)-dependent prostaglandin E2 (PGE2) production has been correlated with inflammation and local estrogen biosynthesis via aromatase. Using a mouse model of 7,12-dimethylbenz[a]anthracene/medroxyprogesterone-acetate (DMBA/MPA)-induced carcinogenesis, we demonstrated that an obesogenic diet promotes mammary tissue inflammation and local estrogen production, and accelerates mammary tumor formation in a COX-2-dependent manner. High-sugar/fat (HSF) diet augmented the levels of the pro-inflammatory mediators MCP-1, IL-6, COX-2, and PGE2 in mammary tissue, and this was accompanied by crown-like structures of breast (CLS-B) formation and aromatase/estrogen upregulation. Treatment with a COX-2 selective inhibitor, etoricoxib, decreased PGE2, IL-6, MCP-1, and CLS-B formation as well as reduced aromatase protein and estrogen levels in the mammary tissue of mice fed a HSF diet. Etoricoxib-treated mice showed increased latency and decreased incidence of mammary tumors, which resulted in prolonged animal survival when compared to HSF diet alone. Inhibition of tumor angiogenesis also seemed to account for the prolonged survival of COX-2 inhibitor-treated animals. In conclusion, obesogenic diet-induced COX-2 is sufficient to trigger inflammation, local estrogen biosynthesis, and mammary tumorigenesis.
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Neoplasias de la Mama/metabolismo , Ciclooxigenasa 2/metabolismo , Dieta Alta en Grasa/efectos adversos , Dinoprostona/biosíntesis , Azúcares/efectos adversos , Regulación hacia Arriba , 9,10-Dimetil-1,2-benzantraceno/efectos adversos , Animales , Aromatasa/metabolismo , Neoplasias de la Mama/inducido químicamente , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Etoricoxib/administración & dosificación , Etoricoxib/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-6/metabolismo , Células MCF-7 , Acetato de Medroxiprogesterona/efectos adversos , RatonesRESUMEN
Glioblastoma multiforme is the most aggressive type of glioma, with limited treatment and poor prognosis. Despite some advances over the last decade, validation of novel and selective antiglioma agents remains a challenge in clinical pharmacology. Prior studies have shown that leguminous lectins may exert various biological effects, including antitumor properties. Accordingly, this study aimed to evaluate the mechanisms underlying the antiglioma activity of ConBr, a lectin extracted from the Canavalia brasiliensis seeds. ConBr at lower concentrations inhibited C6 glioma cell migration while higher levels promoted cell death dependent upon carbohydrate recognition domain (CRD) structure. ConBr increased p38MAPK and JNK and decreased ERK1/2 and Akt phosphorylation. Moreover, ConBr inhibited mTORC1 phosphorylation associated with accumulation of autophagic markers, such as acidic vacuoles and LC3 cleavage. Inhibition of early steps of autophagy with 3-methyl-adenine (3-MA) partially protected whereas the later autophagy inhibitor Chloroquine (CQ) had no protective effect upon ConBr cytotoxicity. ConBr also augmented caspase-3 activation without affecting mitochondrial function. Noteworthy, the caspase-8 inhibitor IETF-fmk attenuated ConBr induced autophagy and C6 glioma cell death. Finally, ConBr did not show cytotoxicity against primary astrocytes, suggesting a selective antiglioma activity. In summary, our results indicate that ConBr requires functional CRD lectin domain to exert antiglioma activity, and its cytotoxicity is associated with MAPKs and Akt pathways modulation and autophagy- and caspase-8- dependent cell death.
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Antineoplásicos/farmacología , Caspasa 8/metabolismo , Activación Enzimática/efectos de los fármacos , Glioma/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Lectinas de Plantas/farmacología , Animales , Apoptosis/efectos de los fármacos , Astrocitos/efectos de los fármacos , Autofagia/efectos de los fármacos , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Glioma/metabolismo , Glioma/patología , Humanos , Ratones , Mitocondrias/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Simulación del Acoplamiento Molecular , Polisacáridos/metabolismo , Dominios Proteicos/fisiología , Estructura Cuaternaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , RatasRESUMEN
The nitroreductase family is comprised of a group of FMN- or FAD-dependent enzymes that are able to metabolize nitrosubstituted compounds using the reducing power of NAD(P)H. These nitroreductases can be found in bacterial species and, to a lesser extent, in eukaryotes. There is little information on the biochemical functions of nitroreductases. Some studies suggest their possible involvement in the oxidative stress response. In the yeast Saccharomyces cerevisiae, two nitroreductase proteins, Frm2p and Hbn1p, have been described. While Frm2p appears to act in the lipid signalling pathway, the function of Hbn1p is completely unknown. In order to elucidate the functions of Frm2p and Hbn1p, we evaluated the sensitivity of yeast strains, proficient and deficient in both oxidative stress proteins, for respiratory competence, antioxidant-enzyme activities, intracellular reactive oxygen species (ROS) production and lipid peroxidation. We found reduced basal activity of superoxide dismutase (SOD), ROS production, lipid peroxidation and petite induction and higher sensitivity to 4-nitroquinoline-oxide (4-NQO) and N-nitrosodiethylamine (NDEA), as well as higher basal activity of catalase (CAT) and glutathione peroxidase (GPx) and reduced glutathione (GSH) content in the single and double mutant strains frm2Delta and frm2Delta hbn1Delta. These strains exhibited less ROS accumulation and lipid peroxidation when exposed to peroxides, H(2)O(2) and t-BOOH. In summary, the Frm1p and Hbn1p nitroreductases influence the response to oxidative stress in S. cerevisae yeast by modulating the GSH contents and antioxidant enzymatic activities, such as SOD, CAT and GPx.
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Nitrorreductasas/metabolismo , Estrés Oxidativo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Antioxidantes/metabolismo , Catalasa/metabolismo , Dietilnitrosamina/metabolismo , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Peroxidación de Lípido , Mutación , Nitrorreductasas/genética , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Superóxido Dismutasa/metabolismoRESUMEN
Diagnosis of Parkinson's disease (PD) remains a challenge in clinical practice, mostly due to lack of peripheral blood markers. Transcriptomic analysis of blood samples has emerged as a potential means to identify biomarkers and gene signatures of PD. In this context, classification algorithms can assist in detecting data patterns such as phenotypes and transcriptional signatures with potential diagnostic application. In this study, we performed gene expression meta-analysis of blood transcriptome from PD and control patients in order to identify a gene-set capable of predicting PD using classification algorithms. We examined microarray data from public repositories and, after systematic review, 4 independent cohorts (GSE6613, GSE57475, GSE72267 and GSE99039) comprising 711 samples (388 idiopathic PD and 323 healthy individuals) were selected. Initially, analysis of differentially expressed genes resulted in minimal overlap among datasets. To circumvent this, we carried out meta-analysis of 17,712 genes across datasets, and calculated weighted mean Hedges' g effect sizes. From the top-100- positive and negative gene effect sizes, algorithms of collinearity recognition and recursive feature elimination were used to generate a 59-gene signature of idiopathic PD. This signature was evaluated by 9 classification algorithms and 4 sample size-adjusted training groups to create 36 models. Of these, 33 showed accuracy higher than the non-information rate, and 2 models built on Support Vector Machine Regression bestowed best accuracy to predict PD and healthy control samples. In summary, the gene meta-analysis followed by machine learning methodology employed herein identified a gene-set capable of accurately predicting idiopathic PD in blood samples.
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Enfermedad de Parkinson , Transcriptoma , Algoritmos , Perfilación de la Expresión Génica , Humanos , Enfermedad de Parkinson/diagnóstico , Enfermedad de Parkinson/genética , Máquina de Vectores de Soporte , Transcriptoma/genéticaRESUMEN
Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumor. Pharmacological treatment of this malignance is limited by the selective permeability of the blood-brain barrier (BBB) and relies on a single drug, temozolomide (TMZ), thus making the discovery of new compounds challenging and urgent. Therefore, aiming to discover new anti-glioma drugs, we developed robust machine learning models for predicting anti-glioma activity and BBB penetration ability of new compounds. Using these models, we prioritized 41 compounds from our in-house library of compounds, for further in vitro testing against three glioma cell lines and astrocytes. Subsequently, the most potent and selective compounds were resynthesized and tested in vivo using an orthotopic glioma model. This approach revealed two lead candidates, 4m and 4n, which efficiently decreased malignant glioma development in mice, probably by inhibiting thioredoxin reductase activity, as shown by our enzymological assays. Moreover, these two compounds did not promote body weight reduction, death of animals, or altered hematological and toxicological markers, making then good candidates for lead optimization as anti-glioma drug candidates.